Restoration of the GTPase activity of G α o mutants by Zn 2+ in GNAO1 encephalopathy models

GNAO1 encephalopathy is a rare pediatric disease characterized by motor dysfunction, developmental delay, and epileptic seizures 1-3 . De novo point mutations in the gene encoding G α o, the major neuronal G protein, lie at the core of this dominant genetic malady 4 . Half of the clinical case mutations fall on codons Gly203, Arg209, or Glu246 near the GTP binding/hydrolysis pocket of G α o 1-3 . We here show that these pathologic mutations strongly speed up GTP uptake and inactivate GTP hydrolysis by G α o, resulting in constitutive GTP binding by the G protein. Molecular dynamics simulations indicate that the mutations cause displacement of Gln205, the key to GTP hydrolysis. Decreased interactions with cellular partners including RGS19 suggest that despite the enhanced GTP residence, the mutants fail to fully adopt the activated conformation and thus transmit the signal. Through a high-throughput screening of approved drugs aiming at correction of this core biochemical dysfunction, we identify zinc pyrithione and Zn 2+ ions as agents restoring the active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with a negligible effect on wild type G α o. We describe a Drosophila model of GNAO1 encephalopathy and show that dietary zinc supplementation restores the motor function and longevity of the mutant �ies. With zinc supplements frequently recommended for diverse human neurological conditions, our work spanning from identi�cation of the core biochemical defect in G α o mutants and cellular interactions analysis to high-throughput screening and animal validation of the de�ciency-correcting drug de�nes the potential therapy for GNAO1 encephalopathy patients. �nally show that the supplementation of the rescues behavioral defects in a Drosophila model of the identifying the potential therapeutic avenue to treat human patients. mutants as the displacement of the catalytic Gln205. The accompanying changes in G α o structure lead to the failure of the G protein to adopt the fully activated conformation by RGS19. Another amino acid residue, T182 playing an important role in the G α -GTP interaction with RGS proteins 23,24 , does not show a signi�cant dislocation in the three pathologic G α o mutants (Supplementary Fig. S4C-E). These observations reveal strong improvements of the behavioral and life span conditions in the Drosophila model of GNAO1 encephalopathy by dietary zinc supplementation. Given the large body of clinical evidence on the dietary zinc supplements for human patients with diverse neurological conditions, our �ndings may speak for the applicability of such supplementation for the pediatric GNAO1 encephalopathy patients. These issues and the sex-sensitive effects of the dietary zinc supplementation are discussed in the Bradford and the and pressure equilibration were performed using typical parameters (50ps duration, 2fs step). A 100ns production run was performed on high performance computation (HPC) cluster of the University of Geneva with 2fs step and leap-frog integrator and with 1nm cutoffs for van der Waals and electrostatic cutoffs. Subsequent analysis of the trajectories and structures was performed using both built-in functions of GROMACS package and PyMol using custom scripts.

R209H was reported to display a faster GTP uptake 12 . We have applied the non-hydrolyzable uorescent BODIPY-GTPγS assay 8,[13][14][15][16][17] to monitor the GTP uptake properties of four Gαo variants: wild type, G203R, R209C, and E246K. This analysis reveals that the three mutants are dramatically faster in uptaking GTP than the wild type (Fig. 1B). The calculated binding rate constant, k bind , increases by the E246K mutation 5-fold, by the R209C mutation -11-fold, and by the G203R mutation -28-fold over that of the wild type Gαo (Fig.   1C).
The faster GTP uptake will lead to the higher GTP residence of the G protein if its GTP hydrolysis rate is not proportionally increased; an accompanying decreased GTP hydrolysis will further aggravate the GTP residence of the G protein. We thus next applied a hydrolyzable uorescent GTP analog, BODIPY-GTP, whose interaction with an active G protein is seen as a transient rise in uorescence (indicative of the nucleotide binding) followed by a decay in uorescence (indicative of GTP hydrolysis due to the lower quantum yield the resultant uorophore-GDP on the protein) 8,15,16,18 . Remarkably, we see that all three pathologic Gαo mutants reveal a dramatically slowed down uorescent decay rate as compared to the wild type (Fig. 1D). Calculation of the hydrolysis rate constant, k hydr , con rms this assessment: k hydr of E246K is reduced 65-fold, of R209C -114-fold, and of G203R -212-fold as compared to the wild type Gαo (Fig. 1E).
Thus, mutations in the three most frequently affected GNAO1 encephalopathy amino acid residues lead to a dramatic increase in the rate of GTP uptake accompanied by a gigantic drop in the rate of GTP hydrolysis. We thus must conclude that biochemically, mutations in Gly203, Arg209, and Glu246 of Gαo lead to the constitutive GTP-binding state of the G protein -the molecular feature likely at the core of the disease etiology.

Defective cellular interactions of the GNAO1 encephalopathy mutants
The distorted proportion of the GTP/GDP-state residence of the GNAO1 encephalopathy mutants inferred from the biochemical experiments must have consequences at the cellular level. We thus moved to express the mutants in mouse neuroblastoma N2a cells, frequently used to study the Gαo function 13 . In this as other cell lines, wild type Gαo shows dual localization at the plasma membrane and the Golgi apparatus, as previously reported by us 13,19 . The G203R, R209C, and E246K mutants show a similar dual localization ( Supplementary Fig. S1), indicative of the normal post-translational modi cations of the mutants. We next tested for the interaction of the G203R mutant -the most disturbed in terms of the GTP/GDP balance as judged by the biochemical experiments above -with its two cellular interaction partners: Gβγ that preferentially interacts with the GDP-loaded form of Gαo, and RGS19 that preferentially binds to the GTP-loaded form 8 . Using an internal GFP fusion (that does not impede the proper localization nor protein-protein interactions 11 ) of Gαo and Gαo[G203R], we rst found in the pull-down assay that the mutation signi cantly reduces the ability of the protein to interact with Gβγ ( Supplementary Fig. S2A, B). This nding agrees with the reduced proportion of the GDP-loaded form of Gαo [G203R], and goes in the same direction as that observed for the classical constitutively activated point mutant, Q205L ( Supplementary Fig. S2A, B).
However, when looking at the interaction with RGS19, we surprisingly found a similar interaction reduction for Gαo[G203R], opposite to that seen for the constitutively activate Q205L ( Supplementary Fig. S2C, D). Puzzled by this nding, we used C-terminally GFPtagged Gαo, this time comparing the RGS19 interactions for the wild type and the three mutant variants of the protein. This analysis again revealed that the GNAO1 encephalopathy mutants display strongly reduced interactions with RGS19 ( Supplementary Fig. S2E, see Supplementary Fig. S7 and Fig. 2 below for representative Western blots). These ndings, put together with the biochemical data, speak for the following. First, given the lack of cellular interaction with the RGS protein whose function is to speed up GTP hydrolysis 7,8 , the cellular GTP-residence of the mutants is expected to be even more signi cant. Second, the mutants display reduced interactions with physiologic Gαo partners and, by inference, reduced signal transduction capacity in neuronal cells. And third, despite the increased residence in the GTP-bound state, the mutant Gαo proteins likely do not adopt the truly activated conformation, hence fail to interact with RGS19 (and presumably other signaling partners).
Displacement of the GTPase catalytic residue in GNAO1 encephalopathy mutants Gαi mutated in the amino acids equivalent to Gαo's Arg209 and Glu246, as well as at the Gly204 neighboring the Gly203, have been proposed to fail to adopt the fully activated conformation 9 , providing a clue to the reduced RGS19 interactions of the Gαo mutants we report above. In order to gain insights into the possible structural de ciency in the G203R, R209C, and E246K encephalopathy mutants that lead to the de cient GTPase reaction and incompetent RGS19 interaction, we performed homology modeling followed by molecular dynamics simulations of the GTP-loaded Gαo, wild type and the mutants, based on the Gαi1-GTPγS structure 20 .
Structural analysis of the energy-minimized state reveals that R209C and E246K, mutations of the amino acids normally forming a salt bridge to fasten switch II and the α3 helix to lock Gα in the active conformation upon the GTP binding 9,21 , result in a signi cant displacement of the Gln205 residue -the key to the catalytic GTPase reaction 22 -away from the γ-phosphate ( Supplementary Fig.   S3A, B, D, also see Fig. 3 below). Through molecular dynamics simulations we further nd a signi cant global destabilization of Gαo[E246K], in agreement with the similar analysis of the Gαi1 structure 9 , but not of the G203R or R209C mutants ( Supplementary   Fig. S4A and Supplementary Movie S1). Further, analysis of energy-minimized model of Gαo[G203R] reveals a somewhat different arrangement as compared to E246K and R209C mutants: instead of acting through switch II, the substituting Arg residue engages in a direct interaction with the catalytic Gln205, displacing the latter from the γ-phosphate and, importantly, occupying the space normally used by the hydrolysis water molecule and thus inducing its displacement ( Supplementary Fig. SC, D, also see Fig. 3

below).
These ndings suggest a common mechanism of the loss of the GTPase reaction in the three GNAO1 encephalopathy mutants as the displacement of the catalytic Gln205. The accompanying changes in Gαo structure likely lead to the failure of the G protein to adopt the fully activated conformation and thus make it poorly recognizable by RGS19. Another amino acid residue, T182 playing an important role in the Gα-GTP interaction with RGS proteins 23,24 , does not show a signi cant dislocation in the three pathologic Gαo mutants ( Supplementary Fig. S4C-E).
High-throughput assay aiming at recovering the GTPase activity of Gαo[E246K] identi ed zinc pyrithione as a drug speci cally acting on the mutant but not wild type proteins We next argued that since the inability of the three Gαo encephalopathy mutant proteins to hydrolyze GTP likely lies at the core of the molecular etiology of the disease and represents an easily measurable biochemical characteristic, one could design an assay to screen for molecules potentially capable of restoring this de ciency. To build such a high-throughput screening (HTS) platform, we employed Gαo[E246K] and BODIPY-GTP to monitor the GTP uptake and hydrolysis by the mutant, as described in the rst section, and screened a library of 2736 FDA-approved and pharmacopeial drugs ( Supplementary Fig. S5A). While wild-type Gαo hydrolyzes all BODIPY-GTP provided to it in the biochemical setting within 10 minutes of the experiment, Gαo[E246K] fails to do so, resulting in the stable BODIPY uorescence (see Fig. 1D). Thus, the initial screening was based on the identi cation of drugs capable of inducing a drop in uorescence by the 10 minutes incubation of Gαo[E246K] with BODIPY-GTP. The screening was followed by hit validation, that resulted in three hit compounds: sennoside A, sennoside B, and zinc pyrithione (ZPT) ( Fig Of those, sennoside A and B were found to decrease GTP uptake by Gαo[E246K], rather than GTP hydrolysis by it ( Supplementary Fig.   S5D, E). In contrast, ZPT restored the GTP hydrolysis (Fig. 2B). When retesting the drugs on wild-type Gαo, sennosides were found to equally affect the GTP uptake by it; in contrast, ZPT revealed the action speci c for Gαo[E246K] but not wild type Gαo ( Fig. 2C and Supplementary Fig. S5F, G), immediately raising our interest to this molecule. Zn 2+ is the active component of zinc pyrithione restoring the GTPase activity of the three encephalopathy Gαo mutants We next found that ZPT revealed a concentration-dependent restoration of the GTPase activity of all the three encephalopathy Gαo mutants: G203R, R209C, and E246K (Supplementary Fig. S6A-C). ZPT is a coordinated complex of pyrithione -a membranepermeable ionophore 25 -and Zn 2+ ( Fig. 2A) and has the primary indication to treat dandruff and seborrheic dermatitis 26 ; other biological activities of ZPT including antifungal, antiviral, and anticancer [27][28][29] have also been reported. Zn 2+ ions are poorly penetrant through cellular membranes 30 , and the pyrithione moiety of the drug serves to deliver the ions inside cells 31 . We thus found that in our cell-free assay monitoring GTP binding and hydrolysis by Gαo, the Zn 2+ ions are active in restoring the GTPase activity ( Supplementary Fig. S6D-F) unlike the 'empty' ionophore (data not shown). We further tested several other metal ions, revealing that none of them recapitulated the effect of Zn 2+ : Co 2+ , Fe 2+ , Ni 2+ , Mn 2+ , and Li + were inactive in restoring the GTPase reaction, while Cu 2+ at 100µM appeared to completely inactivate the G protein ( Supplementary Fig. S6G). In contrast to the ZPT-or Zn 2+ -mediated concentration-dependent restoration of the GTPase activity, the effect of both compounds on wild type Gαo was con rmed to be negligible (Fig. 2D, E). An interesting observation refers to the manner the three mutants respond to ZPT / ZnCl 2 : while restoration of the GTPase reaction in Gαo[R209C] and Gαo[E246K] was saturating with the EC 50 values around 10µM, the response of Gαo [G203R] was rather linear in the concentration range tested (Fig. 2D, E), suggesting the potentially distinct molecular mechanisms of Zn 2+ acting on the G203 mutant vs. the R209C / E246K mutants.
Potential mechanism of action of Zn 2+ in restoring the GTPase activity of GNAO1 encephalopathy mutants We applied structural modeling and molecular dynamics simulations in order to gain insights into the potential mechanism of action of Zn 2+ in the restoration of the GTPase activity of the three Gαo mutants. We argued that Zn 2+ can replace Mg 2+ in the Gαo's active center upon the interaction with GTP: it is well-known that proteins' Mg 2+ -binding sites are generally unspeci c for this ion, which can be substituted by a broad range of other divalent metal ions, with Zn 2+ being one of the most potent substitutes 32 . Our analysis shows that the substitution of Zn 2+ for Mg 2+ in the active center does not affect the global structure in the energy-minimized state of wild type Gαo ( Supplementary Fig. S4B, Supplementary Movie S2). Interestingly, the global rearrangement observed in Gαo [E246K] was to a certain degree further aggravated in the Zn 2+ -bound protein; no global effect of Zn 2+ however, was seen for the G203R and R209C mutants ( Supplementary Fig. S4B, Supplementary Movie S2), suggesting that it is unlikely to represent the general or main mechanism of Zn 2+ action to restore the GTPase activity.
We thus next paid special attention to the position and exibility of the catalytic Gln205 that we found to be placed away from the GTP' γ-phosphate in the pathologic mutants (see Supplementary Fig. S3). Our molecular dynamics simulations reveal that for all three mutant variants, distance between the γ-N atom of Gln205 and the γ-P atom of GTP (increased as compared to the wild type Gαo) is reduced back to the wild type levels by Zn 2+ (Fig. 3). It should be noted that in the wild type protein Zn 2+ also decreases the distance between Q205 and γ-phosphate, but to a considerably smaller extent that apparently does not affect the catalytic activity. Overall, our structural analysis suggests the atomistic mechanism for the action of Zn 2+ on the restoration of the GTPase activity of the E246K and R209C mutants as the bringing back of the catalytic Gln205 to the γ-phosphate of GTP, otherwise swayed away by the mutations. The GTPase restoration mechanism for the G203R mutation may involve the same principle but could additionally employ other features, as hinted at by altered position of the hydrolytic water molecule ( Supplementary Fig. S3C, D) and the different sensitivity of the mutant to ZnCl 2 as compared to the other two mutants (see Fig. 2D, E and the section above).

ZPT restores cellular RGS19 interactions of GNAO1 encephalopathy mutants
Our in vitro data show that ZPT and its ion component, Zn 2+ , are able to restore the GTPase activity of the pathologic Gαo. We next wondered whether such a restoration could be seen in vivo, and be re ected in a restored interaction with a Gαo partner. To this end, we tested if the mutants' interaction with RGS19 can be restored in the N2a cells. It is known, however, that Zn 2+ can either cross the cellular membranes by active transporters 30 or with the help of ionophores such as pyrithione 31 . On the other hand, a signi cant neuronal and other cell toxicity of ZPT has been reported, mainly due to its effectiveness in bringing zinc inside the cells [33][34][35] . Thus, we rst investigated the acute cytotoxicity of ZPT, pyrithione, and ZnCl 2 in N2a cells. This analysis con rms the neurotoxicity of ZPT with the IC 50 of ca. 5µM; ZnCl 2 in contrast was not toxic up to the concentrations of 100µM ( Supplementary Fig. S7A) as presumably it failed to penetrate the cells in this acute setting. For the subsequent experiments on the restoration of the Gαo-RGS19 interactions in N2a cells, we took the highest tested agents' concentrations that did not display cytotoxicity: 1µM for ZPT and 100µM for ZnCl 2 .
Using these concentrations, and performing the experiments with C-terminally GFP-tagged Gαo as in Supplementary Fig. S2E earlier, we found that ZPT could dramatically recover the ability of Gαo[E246K] to interact with RGS19 to the levels normally seen for wild type Gαo ( Supplementary Fig. S7B, C). ZnCl 2 , in contrast, was ineffective. The interaction of Gαo wild-type with RGS19 was also stimulated ca. 2-fold by ZPT ( Supplementary Fig. S7B, C), which is consistent with the certain effect of Zn 2+ on it in molecular dynamics simulation described above or might originate from other, indirect, effects. However, this modest stimulation fades away as compared to the effect of ZPT on the mutant Gαo versions: ca. 6-fold for Gαo[R209C], ca. 12-fold for Gαo [E246K], and the dramatic 20-fold for Gαo[G203R] (Fig. 2E, F). We hypothesize that the ionophore-mediated delivery of Zn 2+ into neuronal cells is capable of restoring the GTPase-competent conformation of the three encephalopathy Gαo variants, the resulting protein-protein interaction(s) and potentially -the signaling functions of the G protein.
Zn 2+ dietary supplementation rescues the Drosophila model of GNAO1 encephalopathy Animal models have the instrumental role in deciphering the human disease mechanisms and in identifying/validating the treatment routes. The fruit y Drosophila melanogaster represents an excellent model organism for studies in various elds of biology 36 , and our recent work highlights the power of Drosophila as the host to model GNAO1 encephalopathy 37 . To establish the GNAO1encephalopathy model in the fruit y, we applied the CRISPR/Cas9-mutagenesis together with phiC31-mediated recombinasemediated cassette exchange 38 to introduce the pathogenic G203R mutation into the Drosophila Gαo (see Methods and Supplementary Fig. S8). We found that the resultant [G203R]/+ ies are viable and fertile yet reveal a number of de ciencies.
Speci cally, the heterozygous mutant ies manifest a signi cant motor dysfunction, measured in the negative geotaxis assay as a reduced capacity to climb up the wall (Fig. 4A, B), reminiscent of the motor dysfunction in the human patients. Further, the encephalopathy mutant Drosophila display a two-fold reduction in the life span (Fig. 4C).
Dietary zinc supplementation has been applied to treat various human health conditions, including the neurological ones such as depression 39 , epilepsy 40 , psychiatric and neurodegenerative diseases 41 , or sleep disorders 42 , as well as for normal neonatal development 43 . We thus wondered if dietary supplementation of zinc in the Drosophila model of GNAO1 encephalopathy may reveal any bene cial effects. Food supplementation of ZnCl 2 to the nal concentration of 200µM has been previously shown to rescue the survival of Drosophila mutant for dZip1 and dZip2, the gut zinc transporters 44 , providing us a guideline. We also tested 10µM ZPT food supplementation, following the application of up to 15µM ZPT in rats 45 . Drosophila lines -G203R/+ and the wild type control (see Methods and Supplementary Fig. S8) -were raised from the egg at the standard food or that supplemented with ZnCl 2 or ZPT.
The climbing capacity of the resultant populations were compared, along with their longevities.
We found that the ZnCl 2 -containing food signi cantly improved the motor function of the G203R/+ ies (Fig. 4D). The effect was particularly strong for female Drosophila, bringing the climbing capacity towards the wild type levels (Fig. 4D). Interestingly, the control wild type ies showed instead a clear reduction in the climbing capacity, except for the ability of ZnCl 2 to modestly improve the climbing of female ies (Fig. 4E). Second, ZnCl 2 food supplementation rescued the reduced life span of female G203R/+ ies (Fig. 4F); no effect could however be seen for ZPT or for male ies (Fig. 4F and Supplementary Fig. S9).
These observations reveal strong improvements of the behavioral and life span conditions in the Drosophila model of GNAO1 encephalopathy by dietary zinc supplementation. Given the large body of clinical evidence on the dietary zinc supplements for human patients with diverse neurological conditions, our ndings may speak for the applicability of such supplementation for the pediatric GNAO1 encephalopathy patients. These issues and the sex-sensitive effects of the dietary zinc supplementation are discussed in the next section. Discussion GNAO1-dependent pediatric encephalopathy is a recently diagnosed rare yet devastating neurological disease. The number of discovered different -mostly missense point -mutations in GNAO1 causing this malady steadily increases every year since the rst 2013' report 4 . However, despite some insights 4,19,46 , the understanding of the molecular etiology underlying the pathological developments has been largely missing. This delay in the understanding blocks development of therapies to treat the patients. Being mostly unresponsive to the conventional anti-epileptic treatments, the sick children have so far demonstrated the best -albeit partial -response to the symptomatic, highly invasive and poorly accessible therapy: the deep-brain stimulation 10 .
The core molecular dysfunction we have "diagnosed" here for the three most common GNAO1-encephalopathy mutations is the constitutive GTP binding state of the mutant Gαo proteins, resulting from a strongly increased rate of GTP uptake concomitant with a dramatically reduced rate of GTP hydrolysis. The failure of the mutants to interact with the GTPase-activating RGS19 is expected to further aggravate this constitutive GTP-bound status of Gαo in GNAO1-encephalopathy neurons. Molecular dynamics and structural modeling provide us with the unifying mechanism of this dysfunction: each of the three mutations induces a displacement of the catalytic Gln205; this change is also likely the reason for the defective interaction with RGS19 -and by inference other physiologic partners of Gαo, thus creating a signaling-de cient G protein.
It is remarkable that a simple ion, Zn 2+ emerging from our screening of drug candidates to recover the GTPase de ciency of the mutant Gαo, is e cient in restoring the structural rearrangements induced by the pathologic mutations. Replacing Mg 2+ from the GTP pocket of Gαo, Zn 2+ brings back the catalytic Gln205 to the vicinity of the γ-phosphate of GTP. This compensation is stronger for the R209C and E246K mutants and weaker for the G203R mutant, suggesting that additional mechanisms may exist for the restoration of the GTPase activity in Gαo[G203R] that would require more direct -NMR or crystallographic -future structure elucidations. The resulting structural rearrangements are su cient to recover the GTPase reaction of the three pathologic mutants, their cellular interactions with RGS19, and ultimately provide motor activity and longevity ameliorations in the Drosophila model of GNAO1encephalopathy upon dietary zinc supplementation.
Dietary zinc supplements have found numerous applications in human health. Being safe, they have been shown to improve neonatal brain development 43 , sleep quality in adults 42 , and to ameliorate health conditions in depression 39 , epilepsy 40 , and a set psychiatric and neurodegenerative states 41 . For example, daily 25mg Zn 2+ applied for 6 weeks in one study 47 , and daily 220mg zinc sulphate (providing 50mg zinc) applied for 12 days in another 48 , was found to positively act on depression patients. The upper limits of dietary zinc with no observed adverse effects, as set by the World Health Organization, are 13 mg/day for the age of 7-12 months, 23 mg/day for the age of 1-6 years, and 45 mg/day for adults 49 . In this regard, dietary zinc supplementation might be considered as a potential treatment option to improve the conditions of GNAO1-encephalopathy patients -at least those carrying the G203R, R209C, and E246K mutations. More studies will show how applicable is the Zn 2+ -restorable GTPase de ciency mechanism to the other GNAO1encephalopathy mutants.
Despite decades of diverse applications of zinc supplements in human health, the details of bioavailability, pharmacokinetics, pharmacodynamics, and potential toxicities of the dietary zinc are still controversial 41,50 . Multiple factors confound the e ciency of zinc absorption from the gut, its penetration through the blood-brain barrier, and the ultimate entry and activities within neuronal cells 41,51,52 . For example, the different e ciency of dietary zinc supplementation in rescuing the motor dysfunction and reduced life span in female vs. male fruit ies ( Fig. 4 and Supplementary Fig. S9) might be related to different expression of certain zinc transporters in the two sexes 53 . Although we consider it unlikely to be re ected in human patients, certain gender differences in the outcome of genetic manipulation of different zinc transporters have been observed in mouse models 54-57 . Diverse approaches to enhance and control zinc uptake and delivery are being developed, from nutritional chelators and nanoparticle carriers to intravenous, cerebrospinal or intra-brain injections 41,52,58 , and might be considered in the future prospective applications to GNAO1encephalopathy patients.
To sum up, the study presented here sweeps from the understanding, at the molecular and even atomistic level, of the core biochemical dysfunction seen in the three most frequent GNAO1-encephalopathy mutations to establishment and screening for drug candidates to rescue this dysfunction, to be followed by the candidate validation in biochemical and cellular models.

Plasmids and molecular cloning
The plasmids for the Gαo-GFP (C-terminally and internally tagged), mRFP-Gβ1, mRFP-Gγ3, and His 6 -RGS19 were previously described 8,11 . The Gαo mutants were obtained by site-directed mutagenesis in the pcDNA3.1 plasmid 8 using the primers as listed in Supplementary Table S1. The plasmid pET-23a encoding wild-type N-terminally tagged 6xHis-Gαo 8 was used to create E246K, R209C and G203R mutants through subcloning using restriction sites SphI and EcoRI from the constructs in pcDNA3.1.

Protein production and puri cation
The Rosetta-gami E. coli strain was transformed with pET23b-Gαo wild type, pET23b-Gαo[G203R], pET23b-Gαo[R209C], or pET23b-Gαo[E246K] and grown at 37°C to OD 600 =0.6 before induction with 1mM IPTG and additional growth overnight at 18°C. Cells then were harvested by centrifugation 3,500g at 4°C and resuspended in lysis/binding buffer containing 50mM TBS, 1mM PMSF, and 30mM imidazole. Cells were disrupted with High-Pressure Cell Press Homogenizer, the debris was removed by centrifugation at 15,000g/ 15min/ 4°C. The supernatant was applied to the Ni 2+ resin (Qiagen) overnight in a rotary shaker at 4°C. The Ni 2+ resin was washed two times with 10 resin volumes of the washing buffer containing 50mM TBS, 1mM PMSF, and 10mM imidazole. On the third wash, the washing buffer was supplemented with 3% glycerol, 10mM MgCl 2 , 0.1mM DTT, and 200μM GDP. The Ni 2+ resin was washed two more times with 10 resin volumes of the washing buffer. Proteins were then eluted with the buffer containing 50mM TBS, 1mM PMSF, and 300mM imidazole. To subsequently remove imidazole, the protein buffer was exchanged into 50mM TBS using Vivaspin concentrator. Protein concentration was measured using the Bradford assay and the purity was analyzed using SDS-PAGE followed by Coomassie staining.

GTP binding and hydrolysis assay
The GTP binding and hydrolysis assay using BODIPY-GTP (Invitrogen) or BODIPY-GTPγS (Invitrogen) was performed as described 8 .
Gαo was diluted to 1μM in the reaction buffer containing 50mM TBS, 10mM MgCl 2 , and 0.5% BSA. The mixture was then pipetted into black 384-well plates (Greiner) and BODIPY-GTP or BODIPY-GTPγS (1μM; Invitrogen) was added into the wells. Fluorescence measurements were performed with a Tecan In nite M200 PRO plate reader with excitation at 485nm and emission at 530nm at 28°C. To trace the fast kinetics of the Gαo[G203R] and Gαo[R209C], the uorescent nucleotide solution was added using the injector unit followed by immediate measurement. The GTP binding and hydrolysis data were t to obtain the k bind and k hydr rate constants as previously described 8 .

Homology modeling and molecular dynamics analysis
The structure of wild-type GTPγS-bound Gαo was homology modeled using the PDB 1GIA structure 20 on the Swiss-Model server with the User Template setting 59 . This structure was used as a base to generate amino acid substitutions in the PyMol software and metal ion substitutions using Check My Metal web interface 60 . The resulting draft PDB models of Gαo mutants bound to Mg 2+ or Zn 2+ were directly used in the GROMACS 2021.2 software 61,62 to generate both the energy-minimized models and the molecular dynamics runs.
To this end, the CHARMM36 all-atom force eld was used. The structures were solvated in a cubic box with 1nm distance from protein edges, the phosphate groups charge was neutralized by Na + ions. Subsequently, energy minimization and temperature and pressure equilibration were performed using typical parameters (50ps duration, 2fs step). A 100ns production run was performed on high performance computation (HPC) cluster of the University of Geneva with 2fs step and leap-frog integrator and with 1nm cutoffs for van der Waals and electrostatic cutoffs. Subsequent analysis of the trajectories and structures was performed using both built-in functions of GROMACS package and PyMol using custom scripts.

High-throughput screening
High-throughput screening (HTS) for mutant Gαo modulators was performed using the Gαo[E246K] protein and FDA Approved & Pharmacopeial Drug Library (HY-L066, MedChemExpress). DMSO or compounds in DMSO (12.5μM) were mixed with Gαo[E246K] at 1μM in a reaction buffer and BODIPY-GTP at 1μM as described in GTP binding assay above. Reaction was carried out for 10min.
To analyze the data generated by the HTS, two parameters were calculated: (i) binding constant (k bind ) and (ii) maximal GTP uptake.
For candidates affecting the k bind , the hits were picked if the compound modulated k bind by ≥2 SD (standard deviation) of DMSOtreated wells. For candidates affecting the maximal BODIPY-GTP uptake, the hits were picked if the compound modulated the maximal GTP uptake by ≥3 SD of DMSO-treated wells. The hits were subsequently validated by performing the GTP binding assay at 50μM of compounds using both Gαo wild type and Gαo[E246K].

Immunoprecipitation (IP)
Immunoprecipitation of Gαo-GFP constructs was performed as previously described 63 . Brie y, N2a cells were transfected with the constructs indicated in the corresponding gures, and after 24h cells were directly harvested or incubated with fresh media supplemented with 1µM ZPT, 100µM ZnCl 2 or DMSO for 3h at normal culture conditions. Cells were harvested with ice-cold GST-lysis buffer (20mM Tris-HCl, pH 8.0, 1% Triton X-100 and 10% glycerol in PBS) supplemented with a protease inhibitor cocktail (Roche). Cell lysates were cleared by centrifugation at 16,000g for 15min at 4°C, and supernatants were incubated with 2µg of nanobody against GFP 64 on ice for 30min. Then, 20μl of a 50% slurry of Glutathione Sepharose 4B beads (GE Healthcare) was added to the samples and incubated overnight on a rotary shaker at 4°C. Beads were repeatedly washed with lysis buffer and bound proteins were eluted by boiling the beads with SDS-PAGE sample buffer. Samples nally were analyzed by SDS-PAGE followed by Western blot using antibodies against GFP (GeneTex GTX113617), His 6 -tag (Qiagen 34650), and mRFP (Santa Cruz sc-101526). Peroxidase conjugated antibodies were from Jackson ImmunoResearch (115-035-062 and 111-035-144). Quanti cation of blots was done using ImageJ from at least 3 independent experiments and statistical analysis was carried out using Student's t-test.

Immuno uorescence and microscopy
For microscopy, N2a cells were transfected for 7h, trypsinized and seeded on poly-L-lysine-coated coverslips in complete MEM for additional 15h before xation. Cells were xed for 20min with 4% paraformaldehyde in PBS, were permeabilized for 1min using icecold PBS supplemented with 0.1% Triton X-100, blocked for 30min with PBS supplemented with 1% BSA, incubated with the primary antibody against GM130 (BD Biosciences 610823) in blocking buffer for 2h at RT, washed and subsequently incubated with the secondary antibody and DAPI in blocking buffer for 2h at RT. The Cy3-labelled secondary antibody was from Jackson ImmunoResearch (115-165-146). Coverslips were nally mounted with Vectashield on microscope slides. Cells were recorded with a Plan-Apochromat 63x/1.4 oil objective on a LSM800 Confocal Microscope and further processed using the ZEN blue software (all Zeiss).

MTT assay
N2a cells (3000 cells/well) were distributed into a transparent 384-well plate. The medium of each well was replaced by 50μl of fresh medium the next day containing the indicated concentrations of ZPT or ZnCl 2 . After incubation for 3h, the medium in each well was replaced by 50μl of 0.5 mg/ml Thiazolyl blue (Carl Roth) solution in 1xPBS. The plates were incubated for 3h at 37°C. Then the solution was removed and 30μl DMSO was added into each well. Absorbance at 570nm was measured in Tecan In nite M200 PRO plate reader.

Plasmids for Drosophila dGαo editing
Donor Plasmid pGao47-LattP-pBacDsRed-attPR for the CRISPR/Cas9 Step of Transgenesis: Plasmid pHD-ScarlessDsRed (Drosophila Genomics Resource Center, Bloomington USA, stock #1364) was modi ed by adding LoxP sequences after dsRed coding region, for which the annealed complementary oligonucleotides GGCCATAACTTCGTATAATGTATGCTATACGAAGTTAT and GGCCATAACTTCGTATAGCATACATTATACGAAGTTAT were cloned into the NotI site of this plasmid. The resultant plasmid (pScarlessDsRed-lox) was digested with AarI and SapI and assembled with two 110bp attP sequences, using NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, cat. #E5520S). Plasmid pTA-attP (Addgene, #18930) was used as a template for PCR ampli cations of attP, which were performed with the primer sets attPfwRI/attPrevHpaI and attPfwKpnI/attPrevHpaI (see the list of primers below). The resultant pattP-pBacDsRed-lox-attP plasmid contains the pBac transposon with the uorescent dsRed marker anked with two inverted attP sequences. The left homologous arm (LHA) was PCR-ampli ed with the dGao47Lfw and dGao47Lrev primers from Drosophila genomic DNA, producing the 815bp PCR product, which was treated with EcoRI and further cloned into the pattP-pBacDsRed-lox-attP plasmid by the EcoRI site producing the construct pLattP-pBacDsRed-lox-attP. The right homologous arm (RHA) was PCR ampli ed with the dGao47Rfw and dGao47Rrev primers, and the resulting 500 bp PCR product was treated with KpnI and then cloned into the plasmid pLattP-pBacDsRed-lox-attP TTCGCTGCTCTTTAATACCGTTA and AACTAACGGTATTAAAGAGCAGC oligonucleotides. The plasmids pDUAL-L1R2 and pDUAL-R1L2 combined with the donor plasmid pGao47-LattP-pBacDsRed-attPR were used for CRISPR/Cas9 step of transgenesis ( Supplementary   Fig. S8).

Locomotion Assay and Measurement of Lifespan of Drosophila
The negative geotaxis assay was performed as described previously 37 . Measurement of lifespan was performed as described 67 ; 110 males and 140 females of each genotype and on each supplemented food were monitored.  It is evident that upon Zn2+ binding, the mutants' Q205 is brought back to the γ-P atom of GTP (the hydrogen bonds are indicated). Color coding as for the respective (A, C, E) panels.